Trpv1 + sensory neurons control of beta-cell stress and islet inflammation in diabetes

ABSTRACT

A process is disclosed for controlling inflammatory tissue access through release of neuropeptides, such as substance P (sP), to insulin-responsive sensory neurons, whereby simultaneous control of insulin sensitivity/resistance is manifested. In models of Type 1 and Type 2 diabetes, sensory afferents, in particular TRPV1, have fundamental roles in insulin/glucose homeostasis, islet physiology and autoimmune tissue inflammation. By manipulation of the TRPV1 neuro-β-cell circuit or enhancement of pancreatic sP levels, normalization of insulin resistance, clearance of inflammation and prevention of both Type 1 and Type 2 diabetes is realized.

FIELD OF THE INVENTION

This invention generally relates to nervous system involvement in themanifestation of insulitis and diabetes; particularly to the mechanismsof the transient receptor potential vanilloid-1 receptor (TRPV1), assuch mechanism relates to clear chronic inflammatory cell infiltrations;and most particularly to the use of Substance P, or similarneuropeptides from sensory afferent neurons, as agents effective for thenormalization of elevated insulin resistance and for the rapidresolution of tissue inflammation and infiltration by immune cells.

BACKGROUND OF THE INVENTION

Type 1 diabetes (T1D) is an autoimmune disease observed in manymammalian species, governed by multiple genetic and environmental riskfactors. Overt diabetes reflects glucose intolerance due to insulindeficiency. It is the end result of prediabetes, with progressivelymphoid infiltration around and then inside pancreatic islets ofLangerhans, and subsequent destruction of insulin-producing β-cells byautoreactive T lymphocytes (Anderson and Bluestone, 2005). T1D ischaracterized by a permissive immune system that fails to imposetolerance to arrays of self-antigens. Although the initiating events arenot fully understood, β-cell stress and β-cell death in the course ofearly islet restructuring are thought to provide sensitizingautoantigens which expand autoreactive T cell pools in pancreatic lymphnodes (Mathis et al., 2001; Rosmalen et al., 2002; Trudeau et al., 2000;Zhang et al., 2002).

Self-antigens targeted in T1D are expressed in β-cells and, in mostcases, elsewhere in the body. They prominently include neuronalantigens, recognized by T cells with pathogenic potential (Salomon etal., 2001; Winer et al., 2001). It is unclear why, in T1D, T cellsinfiltrate only islets and their associated glia (Winer et al., 2003).It is also unclear whether autoimmunity and islet inflammation arerelated to hyperinsulinism and insulin resistance typical for even youngNOD mice (Amrani et al., 1998; Chaparro et al., 2006).

There is evidence for functional interactions between nervous and immunesystems (e.g. (Wang et al., 2003)), but connections between isletautoimmunity and the nervous system remain ill defined (Carrillo et al.,2005). The interface between nervous system, external and tissueenvironments is the primary sensory afferent neuron. Primary afferentsalso have efferent function through local release of mediators such asneuropeptides (e.g. substance P, sP and CGRP). There is evidence thatislets may be innervated by primary sensory neurons, but their localfunction is uncertain (Ahren, 2000).

SUMMARY OF THE INVENTION

With regard to Type I diabetes, it is known that T-cell-mediated deathof pancreatic β-cells leads to insulin deficiency, although themechanism behind what attracts and restricts broadly autoreactivelymphocyte pools to the pancreas remains unclear. The data disclosedherein point to a fundamental role for insulin-responsive TRPV1+ sensoryneurons in β-cell function diabetes pathoetiology.

The instant inventors have now determined that in diabetes-prone NODmice, the role of insulin-responsive TRPV1+ sensory neurons plays a rolein the regulation of both islet autoimmunity and of β-cell stress frominsulin resistance. The instant disclosure demonstrates that eliminatingthese neurons prevents islet inflammation and diabetes, whereassystemic, pathogenic T-cells autoreactivity persists. Additionally, ithas been determined that the insulin resistance and β-cell stress ofprediabetic NOD mice fail to develop when TRPV1+ neurons are eliminated.TRPV1^(NOD) in the Idd4.1 T1D-risk locus, is a hypo-functional mutant,mediating depressed neurogenic inflammation.

It is herein demonstrated that delivering a mediator of neurogenicinflammation, substance P, by intra-arterial injection into the NODpancreas rapidly reverses islet inflammation, abnormal insulinresistance and diabetes for several weeks in animals with sufficientP-cell reserve. Concordantly, TRPV1-knockout significantly enhancesinsulin-sensitivity, while insulitis/diabetes-resistant NODxB6Idd4congenic mice, carrying wild type TRPV1, show restored TRPV1 functionand insulin sensitivity.

Accordingly, it is a primary objective of the instant invention todemonstrate the relationship between the presence of TRPV1+ sensoryafferent neurons, and the manifestation of insulitis and diabetes.

It is a further objective of the instant invention to demonstrate thatelimination of these neurons, in a selective manner, is effective toprevent islet inflammation and diabetes (both Type I and Type II),although systemic, pathogenic T-cell autoreactivity neverthelesspersists.

It is yet another objective of the instant invention to demonstrate thatappropriate delivery of a mediator of neurogenic inflammation, such asSubstance P, is effective, at least transiently, to rapidly reverseislet inflammation, abnormal insulin resistance and Type I and Type IIdiabetes, in subjects with sufficient β-cell reserve.

It is a still further objective of the invention to demonstrate that aninsufficiency in the release of Substance P results in manifestation ofinsulin resistance and the development of both Type I and Type IIdiabetes.

Other objects and advantages of this invention will become apparent fromthe following description taken in conjunction with any accompanyingdrawings wherein are set forth, by way of illustration and example,certain embodiments of this invention. Any drawings contained hereinconstitute a part of this specification and include exemplaryembodiments of the present invention and illustrate various objects andfeatures thereof.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A illustrates, via immuofluorescence, the association of murineislets with meshworks of TRPV1⁺ fibers;

FIG. 1B illustrates that TRPV1 was undetectable in endocrine islet cellsby inmmunofluorescence

FIG. 1C illustrates Western blots in randomly selected 5-6 week oldcontrol and diabetic animals;

FIG. 1D illustrates that in NOD^(caps) mice, islet infiltrations weresignificantly reduced, compared with NOD^(ctrl);

FIG. 1E illustrates that in NOD^(caps) mice most islets remained free oflymphocytes, and there was little insulitis progression over time;

FIG. 1F illustrates that capsaicin treatment delayed the onset ofdiabetes and reduced its incidence;

FIG. 2A-2E compare development and function of systemic T cells inNOD^(caps) and NOD^(ctrl) mice. FIG. 2A particularly shows that systemicT-cell pools autoreactive to islet, pSC and other disease associatedantigens were indistinguishable in NOD^(caps) and NOD^(ctrl) spleencells;

FIG. 2F-2I illustrate the comparative development and function ofpancreatic T cells in NOD^(caps) and NOD^(Ctrl) mice

FIG. 3 illustrates that TRPV1 maps to the Idd4.1 NOD diabetes risksublocus of mouse chromosome 11, and further illustrates the twoin-frame base exchanges characteristic to the NOD sequence;

FIGS. 4A-4F illustrate the results of whole animal experiments in whichTRPV1⁺ sensory afferents were stimulated by subcutaneous capsaicinapplication in NOD and NOR mice;

FIGS. 5A-5F illustrate assessment of TRPV1 function by recordation ofcapsaicin-evoked Ca²⁺ responses in dorsal root ganglion (DRG) neuronsfrom NOD and NOR mice;

FIG. 6A demonstrates the selective delivery of intra-arterially (i.a.)injected Evans Blue dye to pancreatic and pancreatic lymph node tissue;

FIGS. 6B and 6C illustrate that in prediabetic NOD^(ctrl) animals, sPinjection resulted in clearing of islet T cell infiltration;

FIG. 6D illustrates that in animals receiving only pancreatic vehicleinjection, only 6% were lymphocyte free;

FIG. 6E demonstrates that after sP administration, and without insulintherapy, over half of the i.a. injected diabetics normalized bloodglucose levels;

FIG. 6F illustrates that raising pancreatic sP levels enhanced insulinsensitivity;

FIG. 6G illustrates NK1R expression on a portion of T cells frompancreatic lymph nodes;

FIG. 6H demonstrates sP response of activated CD4+ NOD T cells in vitro;and illustrates that substance P abrogated cell proliferation andsurvival in a dose-dependent fashion;

FIG. 6I illustrates that systemic injection of sP did not have a similareffect;

FIG. 7A-7D illustrate differences in glucose and insulin tolerancebetween NOD.scid^(ctrl) versus NOD.scid^(caps) mice;

FIGS. 8A-8D illustrate that TRPV1 plays a fundamental role in isletinflammation and insulin homeostasis, using NOD.B6.idd4-congenic mice;

FIG. 9 illustrates that TRPV1 was undetectable in endocrine cells byRT-PCR of purified NOD islets;

FIG. 10 illustrates that NOD^(ctrl) and NOD.scid pancreas showsaccumulation of more sP in nerve endings that B6 mice;

FIG. 11 illustrates Sjogren like sialitis in both NOD^(ctrl) andNOD^(caps) mice;

FIGS. 12A-12D illustrate NOD^(ctrl) and NOD^(caps) thymocytesubpopulations at 10 days and 6-8 weeks of age;

FIG. 13 illustrates chemically induced delayed hypersensitivity (DTH)responses in NOD^(ctrl) and NOD^(caps) mice;

FIG. 14 demonstrates diabetes transfer efficiency of splenocytes fromNOD^(ctrl) or NOD^(caps) mice when transferred i.v. to untreatedNOD.scid mice;

FIG. 15 illustrates the percentage of regulatory T cells in pancreaticlymph nodes after cyclophosphamide induced Type 1 diabetes;

FIG. 16 illustrates that the DRG levels of the neuropeptide, substance P(sP) were elevated in NOD compared to NOR mice;

FIGS. 17A-17C glucose metabolism in CD-1 mice;

FIG. 18 demonstrates that even in mice that failed to reversehyperglycemia, i.a. sP resulted in significant improvement of metaboliccontrol;

FIG. 19 illustrates the β-cell <-> TRPV1 regulatory circuit in NOD mouseT1D pathoetiology.

DETAILED DESCRIPTION OF THE INVENTION

It is known that a prominent subset of sensory neurons expresses theTransient Receptor Potential Vanilloid-1 (TRPV1) protein, a non-specificcation channel that was first identified as the receptor for capsaicin(Caterina et al., 2000; Prescott and Julius, 2003). It is further knownthat TRPV1⁺ neurons are of importance in proinflammatory reactions(O'Connor et al., 2004), and that islet infiltrating lymphocytes expressreceptors for neuropeptides (Persson-Sjogren et al., 2005). Thus, theinstant inventors set out to investigate the possibility that thesesensory neurons may have a role in T1D.

Results: TRPV1⁺ Sensory Afferents Control Onset of Islet Inflammation &Diabetes

Using immunofluorescence, it was observed that murine islets areassociated with meshworks of TRPV1⁺ fibers (FIG. 1 a). TRPV1 wasundetectable in endocrine islet cells by immunofluorescence (FIG. 1 a,b) and by RT-PCR of purified NOD islets (FIG. 9). based on this evidenceof islet innervation by TRPV1⁺ primary afferent sensory neurons, weinvestigated their possible role in T1D pathogenesis, using neonataltreatment of diabetes-prone NOD mice with capsaicin to permanentlyremove these neurons (Caterina and Julius, 2001; Jancso et al., 1977).Two day-old NOD mice received capsaicin (50 mg/kg, s.c.) or vehicle(NOD^(caps), NOD^(ctrl)). As expected from the voluminous literature,capsaicin-treated mice were viable, fertile, without abnormalities ingrowth or gross tissue structure, including pancreas. We confirmed thelack of TRPV1 expression in NOD^(caps) mice, using immunofluorescence(FIG. 1 a, b, TRPV1 green), Western blots (8, 12 & 20 wk), and standardhot-plate testing in randomly selected 5-6 wk old or in diabetic animals(FIG. 1 c). Consistent with loss of neuropeptide secreting TRPV1+neurons, NOD^(caps) mice showed no sP staining (FIG. 10).

Islet infiltration by hematopoietic inflammatory cells begins by 4-5 wkof age, accumulating at the peri-islet Schwann cell (pSC) border; theautoimmune destruction of the pSC mantle is extensive by 8 wks of age, 2months before the onset of overt diabetes (Winer et al., 2003). InNOD^(caps) mice, islet infiltrations were significantly reduced,compared with NOD^(ctrl) (FIG. 1 d, p<0.0001). More than 70% ofNOD^(caps) islets were free of lymphocytes by age 20 wk, whereas even innon-diabetic NOD^(ctrl) mice of that age very few islets werelymphocyte-free (FIG. 1 d). In one third of NOD^(caps) mice, infiltratedislets were entirely absent, as demonstrated by extensive, serialsectioning. In the remaining two thirds of NOD^(caps) mice, most isletsremained free of lymphocytes, with some degree of inflammation in rareislets, but, strikingly, there was little of the typical insulitisprogression over time (FIG. 1 e). Corresponding to the reduction of Tcell infiltrations, neonatal capsaicin treatment delayed the onset ofdiabetes (p=0.0002) and reduced its incidence (FIG. 1 f, p<0.0001, 35wk, n=52 mice/group, life table analysis). No further NOD^(caps) micedeveloped disease over the next 12 wk, with about 80% reduction in finaldiabetes incidence (p<0.0001, Fisher's exact test).

It was observed that NOD mice spontaneously develop a Sjögren-likedisease (sialitis/lacrimitis) which is under genetic controls separatefrom diabetes (Boulard et al., 2002; Cha et al., 2002). NOD^(caps) miceexhibited the same submandibular lymphocyte infiltrates as untreatedcontrols (FIG. 11). Capsaicin treatment thus causes a dramatic reductionin pancreatic islet inflammation and development of diabetes without ageneralized effect on autoimmune infiltrations elsewhere in the NODmouse.

The observed effects of capsaicin treatment on islet infiltration anddisease development could either reflect a failure to generate isletautoreactive T cell pools, a block of tissue accumulation for relevantimmune cell pools or a change in regulatory mechanisms. Capsaicin wasreported to affect some immune functions in other animal models(Chancellor-Freeland et al., 1995; Helme et al., 1987; Nilsson et al.,1991; Santoni et al., 1996). To investigate the possible effect ofcapsaicin on immune functions or development in the NOD mouse, wecompared systemic (FIG. 2 a-e) and pancreatic T cells (FIG. 2 f-i) inNOD^(caps) and NOD^(ctrl) mice. Systemic T cell pools autoreactive toislet—(Insulin, GAD65), pSC—(GFAP, S100b) and other disease-associatedantigens (HSP60, BSA), were indistinguishable in NOD^(caps) andNOD^(ctrl) spleen cells (FIG. 2 a). To probe the development ofautoreactive T cell pools in NOD^(caps) mice, we measured the peripheralfrequency of a prevalent population of diabetogenic CD8+ T cells thatrecognizes residues 206-214 of islet-specific glucose 6 phosphatasecatalytic subunit protein (IGRP) and its homologous, higher aviditymimotope NRP-V7 (Amrani et al., 2001; Amrani et al., 2000; Anderson etal., 1999; Lieberman et al., 2003; Verdaguer et al., 1997; Verdaguer etal., 1996). The size of the circulating NRP-V7-reactive CD8+ T cell poolwas similar in NOD^(caps) and NOD^(ctrl) spleens (0.25±0.1%, p=0.69)(FIG. 2 d). Lymphoid organ cellularities, T cell development and subsetdistributions were also not different in NOD^(caps) and NOD^(ctrl),comparing splenocytes, axillary lymph nodes and thymus (FIG. 12A-12D).Delayed type hypersensitivity reactions developed normally in NOD^(caps)mice (FIG. 13), suggesting the maintenance of antigen presentation andof effector cell generation (Cua et al., 1995; Morikawa et al., 1993).

In contrast, pancreatic NOD^(caps) lymph node tissue containedsignificantly reduced proportions and absolute numbers of CD8+ and ofactivated CD8+CD69+ effector T lymphocytes, cells critical for isletdestruction (DiLorenzo et al., 1998) (FIG. 2 f, g). As a hallmark ofprediabetes progression, prediabetic NOD mice selectively loseCD4+CD25+, and Foxp3+ regulatory T cell subsets in pancreatic lymph nodetissue (Bluestone and Tang, 2005; Pop et al., 2005). However, NOD^(caps)mice maintained their regulatory T cell compartment in pancreatic lymphnodes beyond 12-16 wk of age (FIG. 2 h, i). Thus, there are significantdifferences in the pancreatic, local immune system of NOD^(caps) andNOD^(ctrl) mice, consistent with the suppression of chronic progressiveislet inflammation in these animals.

Conceivably, undetected abnormalities in the NOD^(caps) immune systemmight have influenced T cell pathogenicity and diabetes development.However, NOD^(caps) animals that did develop disease showed insulitisand spleen cells from these animals transferred T1D with normal kineticsto lymphocyte-free NOD.scid recipients that were not treated withcapsaicin (FIG. 14).

We also compared the ability of splenocytes from randomly selectedNOD^(caps) and NOD^(ctrl) to initiate insulitis in such NOD.scid mice.NOD^(caps) and NOD^(ctrl) splenocytes initiated insulitis equally (FIG.2 c). Moreover, we analyzed BDC2.5 T cell receptor transgenic NOD micetreated with capsaicin (BDC2.5^(caps)) (Ji et al., 1999). Splenocytesfrom these animals similarly transferred T1D to untreated NOD^(ctrl)mice (p>0.1).

Low dose cyclophosphamide accelerates NOD diabetes by multiplemechanisms (Hadaya et al., 2005). Low dose cyclophosphamide accelerateddiabetes development in both NOD^(caps) and NOD^(ctrl) (p=ns, FIG. 2 e)and was associated with reversal of the regulatory T cells accumulationpresent previously in NOD^(caps) vs. NOD^(ctrl) pancreatic lymph nodes(FIG. 15). Thus, NOD^(caps) mice retain the principal ability togenerate diabetogenic T cell pools.

Collectively, our observations separate loss of self-tolerance fromtarget tissue invasion as distinct elements of T1D pathogenesis and theydemonstrate that the NOD^(caps) immune system retains pathogenicpotential. TRPV1⁺ sensory neurons thus appear critical for the immunecell accumulation in the pancreas.

NOD TRPV1 is Polymorphic

The above findings identify an important role of TRPV1+ primary afferentneurons in the initiation and progression of islet inflammation and T1D.TRPV1 maps to the Idd4.1 NOD diabetes-risk sublocus on mouse chromosome11, into an approximately 0.3 cM interval downstream of D11 Ndsl (FIG. 3a) (Grattan et al., 2002; Ivakine et al., 2005; McAleer et al., 1995).

Congenic replacement of the NOD Idd4 locus with the homologous B6genomic interval protects from insulitis and, consequently, diabetes,although splenocytes from these congenic animals transfer both,insulitis and diabetes to NOD.scid mice (Grattan et al., 2002). The NODIdd4 risk locus differs from the homologous genomic region in theinsulitis- and diabetes-resistant NOR strain, that carries nearly 90% ofthe NOD genome, including histocompatibility genes and most other T1Drisk loci (Ivakine et al., 2005; Serreze et al., 1994).

We cloned and sequenced TRPV1 cDNA from NOD and NOR mouse dorsal rootganglia (DRG), and confirmed selected sequence regions in NOD and NORgenomic DNA. The NOR TRPV1 was identical to the published wild type (B6and DBA) sequence, but the NOD sequence has two in-frame base exchanges,leading to predicted P₃₂₂->A₃₂₂ and D₇₃₄->E₇₃₄ amino acid replacements(FIG. 3). Both replacements fall into regions highly conserved amongdiverse species (FIG. 3).

TRPV1^(NOD) is Dysfunctional

Further investigation allowed us to determine whether the sequencedifferences in TRPV1^(NOD) might cause abnormalities of TRPV1 function.The innervation of skin by TRPV1+ sensory afferents allowed assessmentof potential functional differences by whole-animal experiments in whichthese afferents were stimulated by cutaneous capsaicin application (FIG.4). Before testing capsaicin we found that there were no differencesbetween NOD and NOR mice in basal withdrawal responses to heatstimulation of the paw or tail (FIG. 4 a), indicating that there was nogeneralized alteration of basal nociception in NOD mice. In addition,the sensitization of heat-evoked withdrawal responses followingintradermal capsaicin administration was not different in NOD versus NORmice (FIG. 4 b). However, nociceptive behavioral responses (biting,licking) evoked by intradermal capsaicin were markedly depressed in NODas compared with NOR mice (p<0.05, FIG. 4 c). Similarly, the paw edemaproduced by capsaicin was significantly reduced in NOD mice (p<0.01,FIG. 4 d), suggesting reduced neuropeptide secretion and inflammation atthe site of stimulation.

The depressed NOD acute nociceptive and neurogenic inflammatoryresponses were not due to ongoing autoimmune inflammation, sinceNOD.scid mice, which lack lymphocytes, were not different from NOD (FIG.4 e, f). Thus, the TRPV1^(NOD) sequence abnormality appears to producedysfunction of TRPV1-mediated responses to capsaicin.

To assess TRPV1 function more directly, we recorded capsaicin-evokedCa²⁺ responses in dorsal root ganglion (DRG) neurons from NOD and NORmice (FIG. 5). The maximum NOD DRG Ca²⁺ response to capsaicin wassignificantly smaller than that of NOR DRG neurons (p<0.01, FIG. 5 a-c).In addition, the maximum capsaicin response was reduced and required10-fold higher drug concentrations in NOD DRG neurons, compared withthat in NOR (p<0.05, FIG. 5 c). In contrast, KCl-evoked Ca²⁺ responsesof NOD and NOR DRG neurons were not different (FIG. 5 d), indicatingthat NOD mice do not exhibit a general abnormality in Ca²⁺responsiveness.

The most direct readout of TRPV1 function are stimulus-evoked currentresponses, and we found that capsaicin-evoked whole-cell currents weresignificantly smaller in DRG neurons from NOD mice as compared with NORmice (FIG. 5 e).

Because of the depressed TRPV1 function, we measured TRPV1 proteinexpression in DRGs and found that the basal TRPV1 protein level in NODmice was lower than that in NOR (FIG. 5 f). Thus, the depression ofcapsaicin-evoked Ca²⁺ and current responses in DRG neurons from NOD micemay in part reflect decreased steady-state expression levels ofTRPV1^(NOD). The right-ward shift in the capsaicinconcentration-response relationship suggests that the functionality ofthe TRPV1^(NOD) protein itself may also be reduced as compared withTRPV1^(wild type). Collectively, we discovered functional abnormalitiesin nociceptive behavior, neurogenic inflammation, channel function andexpression which define TRPV1^(NOD) as a hypo-functional mutant.

Localized Pancreatic Substance P Administration Reverses Islet Pathology

We reasoned that abnormal TRPV1 function might selectively lead to isletpathology, if there was a local, disease-predisposing TRPV1 effect onβ-cell function, and if that effect was removed in NOD^(caps) mice. Theinsulin-rich islet milieu represents a unique environment for TRPV1+nerve terminals, as they express insulin receptors and insulinsensitizes and lowers the activation threshold of TRPV1 channels (VanBuren et al., 2005). Based on the diminished capsaicin-evoked neurogenicinflammation in NOD mice, and the reduced TRPV1 expression and function,we hypothesized that release of mediators of neurogenic inflammationfrom the peripheral terminals of sensory neurons may be depressed inthese mice. One of the principal mediators of neurogenic inflammation isthe neuropeptide, substance P (sP) (O'Connor et al., 2004), andconsistent with reduced release we found that sP levels were elevated inNOD compared with NOR dorsal root ganglia, the location of substance Psynthesis (FIG. 16). NOD^(ctrl) pancreas shows accumulation of more sPin nerve endings than B6 mice and this is not due to inflammation as itwas also observed in NOD.scid mice (FIG. Sx). The enhanced accumulationof sP is consistent with reduced sP release.

If depressed sP release was critical for NOD islet pathology, thenincreasing pancreatic sP levels is predicted to relieve the pathogenicprocess. We therefore injected sP via the pancreatic artery. FIG. 6 ademonstrates the pancreatic delivery of intra-arterially (i.a.) injectedEvans Blue dye, including a pancreatic lymph node (insert). Inprediabetic NOD animals, 12 wk of age, we found that within 2 d afteri.a. sP injection (2 nmoles/kg), about 80% of all islets were free of Tcell infiltration (FIG. 6 b, c), and there were only small, residualinfiltrates in the remainder. Systemic (i.v.) injection of the same sPdose did not have similar effects (see below, FIG. 6 i). In animalsreceiving pancreatic i.a. vehicle injection, only 6% of islets werelymphocyte free (p<0.0001) (FIG. 6 d).

Analogous observations were made following pancreatic i.a. injection ofsP into newly diabetic NOD mice, 2-3 d after diagnosis. Following sPadministration, and without insulin therapy, over half of the i.a.injected diabetics normalized blood glucose levels (FIG. 6 e, redlines). In these responding mice, fasting blood glucose returned to nearnormal levels rapidly and remained at these levels for 2-8 wk. Raisingpancreatic sP levels dramatically enhanced insulin sensitivity,suggesting that the elevated insulin resistance at diagnosis wasnormalized (FIG. 6 f). On average, mice that reversed diabetes had lessextreme hyperglycemia at the time of diagnosis than did thenon-responding mice, likely reflective of a larger residual P-cell massat the time of sP administration. However, even in mice that failed toreverse hyperglycemia (blue lines), i.a. sP caused a significantimprovement of metabolic control, preventing the progressive loss ofbody weight typical of overtly diabetic NOD mice (FIG. 18). Thisimprovement corresponds to significantly (p<0.0001) improved insulinsensitivity (FIG. 6 f, blue line) which enhances the effectiveness of asmall remaining b-cell mass at diabetes onset. In all vehicle-injectedcontrol animals, blood glucose rose progressively, body weights declinedand animals were sacrificed because of severe diabetes between days12-16.

Abundant expression of the NK1R sP receptor has been reported for isletinfiltrating lymphocytes (Persson-Sjogren et al., 2005), and, therefore,one likely target for sP is activated pancreatic T cells. We detectedNK1R expression on a portion of T cells from pancreatic lymph nodes(FIG. 6 g), however, upon in vitro activation with Con A, essentiallyall NOD splenic T cells expressed NK1R (FIG. 6 g, insert). To determinethe functional effect of NK1R ligation, we tested the sP response ofactivated CD4+NOD T cells in vitro. Substance P abrogated cellproliferation and survival in a dose-dependent fashion (FIG. 6 h).

To determine the in vivo effect of pancreatic i.a. sP injection onclonal T cell expansion in pancreatic lymph node, we used isletreactive, BDC2.5 T cell receptor transgenic T cells after labeling withthe fluorescent dye, CFSE (Ji et al., 1999). Cells were transferred into12 wk-old, normoglycemic NOD females which had received pancreatic i.a.or systemic i.v. sP (red lines) or vehicle injections 12-16 hr prior(FIG. 6 i). BDC2.5 T cells from pancreatic lymph nodes were analyzed byflow cytometry 4 days later. Injection of sP reduced cellularity andclonal expansion, measured by dye dilution (p=0.003). Systemic (i.v.)injection of the same sP dose had no effect on expansion of BDC2.5 Tcells in pancreatic lymph nodes, suggesting a pancreastissue-conditioning effect of i.a. pancreas injection that lasts atleast 12-16 hr. A third set of animals received BDC2.5 cells that werein vitro pretreated with sP or vehicle overnight. This in vitropretreatment with sP, reduced the ability of these cells to expand inpancreatic lymph nodes (6i, bottom panel, p=0.0045). As equal numbers ofviable cells were transferred, these observations imply that sP also hasa T cell conditioning effect.

Taken together, the data suggest that reduced neuropeptide release bypancreatic TRPV1+ nerve terminals is a pathogenic event in NOD diabetes,amenable to therapeutic correction.

TRPV1 Function and B-Cell Stress

1-cell stress has previously been suggested as an early element of T1Dpathoetiology, thus an additional objective of this work became thedetermination of whether the hypofunctional TRPV1^(NOD) is related tosigns of b-cell stress, hyperinsulinism and abnormal glucose clearance,observed even in young NOD mice (Rosmalen et al., 2000; van de Wall etal., 2005). We compared measures of β-cell function in untreated and incapsaicin treated NOD.scid mice, and in C57/BL6J (‘B6’) andB6.TRPV1^(−/−) mice, the latter with a normal complement of sensoryafferent neurons but absent TRPV1 expression (Caterina et al., 2000).NOD.scid mice were used to ascertain absence of lymphoid isletinfiltrations in NOD experiments, CD1 mice provided controls (FIGS.17A-17C).

The high-normal serum glucose levels after i.p. glucose challenge in10-12 wk old NOD.scid^(ctrl) mice were significantly reduced inNOD.scid^(caps) mice (FIG. 7 a, p=0.004). The improved NOD.scid^(caps)glucose response was associated with significantly less insulinproduction (FIG. 7 a, insert), suggesting more effective insulin actionfollowing removal of TRPV1⁺ sensory neurons.

B6 mice develop elevated insulin resistance and type 2 diabetes-likedisease (Parekh et al., 1998), attributed to the functional deletion ofnicotinamide transhydrogenase (Freeman et al., 2006). Consistently, weobserved high blood glucose levels after standard i.p. glucose challenge(FIG. 7 b). B6.TRPV1^(−/−) mice showed a significantly improved glucoseresponse, analogous to NOD^(caps) mice, further pointing to thepossibility that TRPV1 may play a general role in β-cell physiology.

To more directly assess if both data sets could reflect enhanced insulinsensitivity due to TRPV1 removal, we measured glucose clearance after asingle insulin injection. Compared to their respective control animals,NOD^(caps) and B6.TRPV1^(−/−) mice showed significantly enhanced andaccelerated glucose clearance, which we interpreted as evidence forreduced insulin resistance due to the absence of TRPV1 in these twoindependent animal models. Similar outcomes in NOD^(caps) andB6.TRPV1^(−/−) mice link the observed effects on β-cell function toTRPV1. Enhanced insulin resistance associated with TRPV1^(NOD)constitutes a persistent β-cell stress, likely worsening withprogressive islet inflammation (Nielsen et al., 2004). TRPV1 and TRPV1+sensory neurons impact insulin homeostasis in these models of Type 1 andType 2 diabetes.

Congenic Replacement of NOD.Idd4

As a test of our conclusions that TRPV1 plays a fundamental role inislet inflammation and insulin homeostasis, we investigatedNOD.B6.Idd4-congenic mice (FIG. 8). These mice carry wild type TRPV1 inthis locus, and are insulitis- and diabetes resistant despite the factthat their splenocytes transfer diabetes to NOD.scid mice (Grattan etal., 2002). Consistent with wild type TRPV1 function, we found thatthese congenics have normalized behavioral responses to cutaneouscapsaicin injection (biting/licking, FIG. 8 a), neurogenic inflammationfollowing paw injection with capsaicin (FIG. 8 b) and Ca²⁺ responses incapsaicin-stimulated DRG (FIG. 8 c). The glucose responses of weanedIdd4 congenics were comparable to those in NOD control mice, however,there was a significant reduction in glucose-induced insulin secretion,suggesting an absence of elevated insulin resistance in these animals(FIG. 8 d).

These NOD congenic mice resemble NOD^(caps) mice, as both areinsulitis/diabetes protected, although their T cells transfer diabetesto NOD.scid recipients. TRPV1^(NOD) adds elevated insulin resistance asnew, strikingly diabetes-relevant phenotype to the NOD Idd4.1 risklocus, presently associated only with insulitis; transgenic rescueexperiments will be required for formal proof that TRPV1 is or is notthe Idd4.1 diabetes risk gene.

Experimental Methods Mice

NOD, NOD.scid, BDC2.5 TCR transgenic NOD mice (‘BDC2.5-NOD’),NOD-β2m^(null), C57BL/6 (‘B6’), B6-TRPV1^(null), NOR NODxB6 Idd4congenic mice (NOD.B6-(D11Nds1-D11Mit325)/DelJ) were obtained from theJackson Laboratories (Bar Harbor, Me.) and maintained under approvedprotocols in our vivarium (NOD female diabetes incidence: 85-90%). Forremoval of TRPV1⁺ neurons, 50 mg/kg capsaicin (20 μL, Sigma, St. Louis,Mo.) was subcutaneously injected into 2 d-old mice with no signs ofadverse effects (NOD^(caps)). Control mice (NOD^(ctrl)) received 20 μLvehicle (10% ethanol, 10% Tween, 80% saline). In adoptive transferexperiments, splenocytes from 4-6 diabetic NOD females were pooled and10⁷ cells/mouse were injected (100 μL i.v.) into irradiated (300 rad) 6-to 8 wk-old NOD.scid recipients. Diastix strips were used to screen forglucosuria (Bayer HealthCare), diabetes was confirmed by diabetic bloodglucose measurements on 2 consecutive days (>13.8 mM/l; SureStep, LifeTechnologies Inc., Burnaby, British Columbia, Canada).

Delayed type hypersensitivity (DTH): DTH responses were elicited aftersensitizing 6 wk old mice with 7% TNCB in 4:1(vol/vol) acetone/oliveoil. Abdomens were shaved and 100 μl of the allergen were applied. 6 dlater, the baseline thickness of right ears were measured by gaugebefore application of 10 μl 1% TNCB in oil or carrier only. Earthickness was re-measured in sensitized and naïve animals 24 hr later.

T-Cell Studies

Spienocytes from 3- to 24 wk-old NOD females were cultured (4×10⁵cells/well) in AIM V serum-free medium (Life Technologies) containingantigen (0.002-50 μg/ml), baculovirus-derived human GAD65 (DiamydDiagnostics, Stockholm, Sweden), human GFAP (>90% pure) and bovine S100b(>98% pure; Calbiochem, San Diego, Calif.) were purchased, otherantigens were previously generated and described (Winer et al., 2003).After 72 hrs, cultures were pulsed (1 μCi, [3H]thymidine/18 hrs) andcounted by liquid scintillation. Lymph node assays were similar, butused 2×10⁵ lymph node cells plus 2×10⁵ irradiated (1,100 rad), syngeneicsplenocytes/well. To normalize pooled data, we calculated a stimulationindex (SI, cpm antigen stimulated/medium control), background countswere <1500 cpm in spleen cell and <1000 cpm in lymph node cultures. Insome proliferation studies, plate-bound anti-CD3 (0.001-3 μg/ml; BDPharMingen) and anti-CD28 (0.2 μg/ml; BD PharMingen) were used tostimulate CD4+T-cells negatively selected from NOD β2 m^(null)splenocytes in the presence of substance P.

Intra-Arterial Pancreas Injection

Animals were anaesthetized with isoflurane and the aorta developed withminimal trauma to ligate above and below the celiac artery. A 32G needlewas used to inject Evans Blue (3 mg/kg/100 μl; Sigma), substance P (2nmol/100 μl; Sigma) or saline into the aorta just prior to the celiacbranching. Ligations are released after closure of the injection site.For insulitis studies, pancreata from treated mice were obtained 48-72hr after pancreas injection treatment and histology performed. Indiabetes reversal experiments, newly diagnosed NOD female mice weretreated with either saline or substance P and followed without exogenousinsulin treatment.

5- and 6-Carboxyl-Fluorescien Succimidyl Ester (CFSE) Labeling

For dye dilution in vivo clonal expansion studies, splenic CD4+ T-cellsfrom NOD-BDC2.5 females were isolated by negative selection (StemcellTechnologies, Vancouver, Canada) and incubated with 2.5 μM CFSE (10′/37°C., Molecular Probes, Eugene, Oreg.) in PBS. Prediabetic (12 wk) NODfemales pretreated 12 hr prior with substance P or saline, were injectedi.v. with 3×10⁶ CFSE-labeled CD4+ T-cells in sterile PBS.

Immunofluorescence and Histology

Frozen murine pancreas sections were fixed in 4% paraformaldehyde,blocked with 5% normal donkey serum (Jackson), and stained withpolyclonal rat or goat antibodies against GFAP (Signet PathologySystems, Dedham, Mass.), polyclonal rabbit anti-TRPV1 (Oncogene) orguinea-pig antibody against insulin (DAKO, Carpinteria, Calif.). Boundantibodies were detected with biotinylated donkey anti-guinea pig or ratIgG (1:200, Jackson), Streptavidin AlexaFluor 546 or 633 (1:300,Molecular Probes, Eugene, Oreg.), and FITC conjugated donkey anti-rabbitIgG (1:25, Jackson). When the biotin-streptavidin system was used,sections were also blocked using an avidin/biotin blocking kit (Vector,Burlingame, Calif.). TRPV1 staining was performed on snap-frozensections of NOD female pancreas with an overnight incubation of primaryantibody at 4° C. To score insulitis severity, pancreata were fixed in10% buffered formalin for a minimum of 24 hr. Histological sections werestained with hematoxylin and eosin and three blinded observers scored atthe following scale: 0, normal islet; 1, peri-insulitis or encroachmentof <25% of the islet surface area; 2, infiltration of 25-50% of theislet surface area; 3, infiltration of >50% of the islet surface area(Winer et al., 2003). Spleen and lymph node cells were stained witheither 2 nM PE-conjugated NRP-V7/H-2 K^(d) or TUM/H-2 K^(d) tetramers,the latter a negative control, in FACS buffer (1% v/v FBS, 0.1% w/v NaN₃in PBS) at 4° C. for 1 hr, followed by staining with FITC-conjugatedanti-CD8 mAb (clone 53-6.7; 5 μg/μL) and PerCP-conjugated anti-B220 Ab(clone RA6-3B2; 2 μg/μL, both from BD Pharmingen). Tetramer positivitywas analyzed on gated CD8+B220-cells and reported as percentage of cellsbinding the NRP-V7/H-2 K^(d) tetramer minus the percentage of cellsbinding the negative control, TUM/H-2 K^(d) tetramer.

Flow Cytometry

Lymphocytes from thymus, pancreatic/axillary lymph nodes and spleen werestained with FITC, PE, and APC conjugated antibodies to CD3, CD4, CD8,CD44, CD25, CD69 CD62L and FoxP3 (BD Pharmingen, not all combinationsare shown). NK-1R and Vβ4 antibodies were obtained from NovusBiologicals (Littleton, Colo.) and Cedarlane (Hornby, Canada),respectively. Live events were collected based on forward- andside-scatter profiles on a FACScan flow cytometer (BD) and analyzedusing FlowJo software (Stanford University).

Molecular Cloning

PCR amplification was performed with cDNA from NOD, NOR, and B6 dorsalroot ganglia using TRPV1 specific primers. Forward:^(5′)ATGGAGAAATGGGCTAGCTTAG^(3′), reverse:^(5′)TCATTTCTCCCCTGGGGCCATGG^(3′). We cloned TRPV1 cDNA using theTOPO®XL PCR Cloning Kit (Invitrogen, Mississauga, ON). Genomic fragmentsof polymorphic TRPV1 regions were cloned using genomic DNA and thefollowing primers: ^(5′)ATGGAGAAATGGGCTAGCTTAG^(3′),^(5′)TGTTGTCAGCTGTGTTATCTGCC^(3′), ^(5′)TTCAGCCATCGCAAGGAGTA^(3′), and^(5′)TCATTTCTCCCCTGGGGCCATGG³, 3-5 independent clones from each mousestrain were sequenced with reproducible results.

RT-PCR: Trizol (Sigma) was used for mRNA extraction from tissues. RTreactions used SuperScript™II RnaseH-reverse transcriptase (Invitrogen):forward

primer: 5′-GGAGAAATGGGCTAGCTTAG-3′, reverse primer:5′-GAAGACCTCAGCATCCTCTGG (XL-PCR Applied Biosystems)

Behavioral Studies and Paw Volume Measurement

Male NOD, NOR, NOD.scid and NOD.B6.Idd4-congenic mice, 5-10 wk old, wereused for behavioral and paw volume measurements. All behavioral testswere conducted between 9:00 and 16:00 hr. Before testing, mice wereallowed to acclimatize to the testing environment for 30 minutes and tothe testing apparatus for 1 hr. Paw thermal withdrawal thresholds weremeasured with a paw thermal stimulator system (UCLA, San Diego, Calif.).The stimulus current was maintained at 4.8 amperes while a 24 secondcut-off was used to limit possible tissue damage. The tail-flick assaywas conducted using a tail-flick analgesia meter (Columbus Instruments).Paw volumes were measured using a commercially available plethysmometer(Ugo Basile) and values were standardized by expression as a percentageof individual preinjection volumes, to accommodate the variation in bodyweights. Capsaicin (0.1 μg/10 μl) was injected s.c. into the plantarpart of mouse hind paw. Capsaicin induced biting/licking time wasrecorded for the first 5 minutes. Paw withdrawal thresholds weremeasured 15 min following capsaicin, and paw volume was measured 45 minfollowing capsaicin injection. NOD^(caps) and NOD^(ctrl)thermosensitivity was analyzed by standard heated (56° C.) plate assay,measuring time to biting/licking response.

Ca²⁺ Response Measurement

Dorsal root ganglia from male NOD, NOR mice and NOD.B6.Idd4-congenic(5-10 wk) were isolated and cultured in F12 medium (Invitrogen) with 10ng/mL nerve- and 10 ng/mL glial-derived nerve growth factor. Cultureswere used 3-5 d after plating. The Ca²⁺-sensitive fluorophore, fura-2(Molecular Probes, Eugene, Oreg.), was used to assess [Ca²⁺]_(i) byratiometric measurement. Excitation (340 and 380 nm) was generated by axenon arc lamp and passed through a high-speed, computer-controlled,variable-wavelength monochromator. This light was transmitted to therecording dish via a fiberoptic cable. Emitted light was directedthrough a 510 nm bandpass filter and detected by an intensified CCDcamera. Image data were analyzed off-line. Each 340 nm image wasdivided, on a pixel-by-pixel basis, by the corresponding 380 nm image,producing a ratio. Averaged values of the ratios within each region ofinterest were plotted as a function of time.

Electrophysiological Recording of TPRV1 Currents

Whole-cell patch-clamp recordings were performed 3-5 d after preparationof DRG neurons. Standard bath solution contained (in mM): 140 NaCl, 5KCl, 2 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4 (adjusted with NaOH).Pipette solution contained (in mM): 140 CsF, 10 BAPTA, 1 CaCl₂, 2 MgCl₂,10 HEPES and 4 K₂ATP, pH 7.3, osmolarity 300 mosM. All patch-clampexperiments were performed at room temperature. TRPV1 currents wererecorded using an Axopatch 1-D amplifier, data were digitized withDigiData 1322, filtered (2 kHz), and acquired by the pClamp9.0 program.Recordings in which the series resistance varied by more than 10% wererejected.

Immunoblotting

DRG or dorsal horns (spinal cord) were dissected and immediately frozenat −80° C. Upon thawing, they were homogenized in lysis buffer (in mM:20 Tris, pH8, 137 NaCl, 2 EDTA, 1 sodium vanadate, 5 NaF, 1phenylmethanesulfonyl fluoride (PMSF), glycerol 10%, Nonidet P-40 1%,SDS 1%, anti-pain 10 μg/mL, leupeptin 10 μg/mL, pepstatin 10 μg/mL, allSigma) at 4° C. Total protein (45 μg DRG protein, 30 μg spinal cord)were electrophoresed (10% acrylamide gels), western blotted, probedovernight with rabbit anti-TRPV1 antibody (1:250, Oncogene) anddeveloped with the ECL kit (Amersham). As a loading control for eachlane, membranes were stripped and reprobed with mouse anti-β actinantibody (1:4000, Sigma). Densitometric analysis employed NIH Image-Jsoftware.

Statistics

All tests were 2-tailed, significance was set at 5%. Life tables,t-tests (flow cytometry), ANOVA and Fisher's exact test were used asdescribed in the text.

CONCLUSIONS

In summary, in the different, independent animal strains andexperimental conditions analyzed, TRPV1 emerges as a central controllerof both islet stress and T cell infiltration. Elimination of TRPV1+neurons by capsaicin, transient functional normalization by acute localsP injection or replacement with wild type TRPV1 in Idd4 congenics allhave the same, islet-specific outcomes: normalized insulin sensitivityand abrogation of insulitis, despite unimpeded generation ofautoreactive lymphocytes that can transfer disease to untreated NODhosts.

One explanation which unifies these observations is a local feedbackinteraction between β-cells and the primary sensory neurons innervatingthe islets (FIG. 19). Such a mechanism has been proposed previouslybased on more indirect evidence (Hermansen and Ahren, 1990). Normally,this interaction is in balance, but in the NOD mouse, hypofunction ofTRPV1 unbalances the feedback, leading to β-cell stress and infiltrationby autoreactive T cell pools independently generated in the NOD mouse.Removing TRPV1 neurons leads to elimination of the unbalanced,pathogenic interaction, while administering sP exogenously appears tore-normalize the interaction, at least transiently.

With specific reference to FIG. 19, islet insulin ligates insulinreceptors on TRPV1+ sensory afferent islet terminals which lowers theactivation threshold of TRPV1 with subsequent Ca²⁺ influx and localrelease of neuropeptides (e.g. substance P, CGRP). Secreted mediatorsinsulin production in a tonic balance. Our observations indicate thatthis balanced feedback loop is essential for normal β-cell homeostasis.

Neonatal capsaicin treatment suppresses NOD islet infiltration and localexpansion of diabetogenic T-cells without detectable impairment ofglobal T-cell function, including typical NOD autoreactivity. Thetreatment normalizes β-cell stress as measured through insulinsensitivity and glucose responses. We have mapped this effect to theTRPV1^(NOD) gene in the Idd4 diabetes risk region, and shown that thatTRPV1^(NOD) is a hypo-functional mutant with considerable reduction inTRPV1 signaling, expression and downstream release of neuropeptides.Focusing on one major neuropeptide secreted by TRPV1+ neurons, sP, wedemonstrate that sP has a direct deleterious effect on T cells, mostexpressing detectable NK1R sP receptors following activation.

A direct neuropeptide effect for β-cells has previously been reported,with deleterious outcomes at low concentrations, but β-cell augmentingeffects at higher concentrations (Barakat et al., 1994; Bretherton-Wattet al., 1992; Hermansen and Ahren, 1990). Our hypothesis, based on theforegoing, that in NOD mice suppressed neuropeptide secretion is apathogenic event, was positively answered through two independentapproaches: removal of TRPV1+ neurons and local i.a. pancreas injectionwith sP, both of which evidenced similar results.

Pancreas sP injection normalized all parameters tested: clearing ofinsulitis lesions, enhancement of insulin sensitivity and consequentreversal of overt diabetes that lasted for a period of weeks. Thismethodology is in marked contrast to the only other strategy to reverseNOD diabetes, which is toxic immunosuppression with anti-CD3 antibodies,now also in clinical trials with human diabetics (Keymeulen et al.,2005).

When viewed in their totality, our findings are inconsistent with theview that diabetes is due solely to immunological and endocrineabnormalities. Rather, our observations demonstrate that the nervoussystem, in particular TRPV1⁺ primary afferent neurons, have a criticalrole in diabetes pathoetiology. Analogous findings in NOD^(caps),NOD.Idd4 congenics and in TRPV1 knockout mice add strength to ourconclusions, as does an earlier report demonstrating that anotherTRPV1-dependent neuropeptide, CGRP, prevents diabetes whentransgenically overexpressed in the islet (Khachatryan et al., 1997).

We have recently generated preliminary evidence for insulitis anddiabetes protection by selective trans-section of sensory nervesinnervating the pancreas, providing yet another line of support for therole of TRPV1+ sensory neurons in T1D pathoetiology.

The mapping of several NOD disease-associated phenotypes to a single,mutant protein, TRPV1, implies that TRPV1⁺ sensory afferents are keyelements for normal islet physiology, opening broad new areas ofresearch including insulin resistance, which remains a challenge afterdecades of intense investigation (LeRoith and Gavrilova, 2006), thatrecently has included sensory nervous system elements (Moesgaard et al.,2005).

The data generated to date has enabled us to identify the molecularmechanism that translates a system-wide genetic TRPV1 defect intopancreas-specific disease. TRPV1+ sensory neurons express high affinityinsulin receptors, insulin potentiates TRPV1 currents (Van Buren et al.,2005), and lowers TRPV1 thermal activation thresholds (Sathianathan etal., 2003). At body temperature, the insulin-rich islet milieu shouldgenerate tonic TRPV1 current activation with associated neuropeptiderelease impacting on basal insulin secretion, a local control circuitfirst envisioned over a decade ago (Hermansen and Ahren, 1990). In NODmice, this sensory nerve terminal-β-cell circuit has gone astray, withdisease prevention through either its removal, or through sufficientlocalized supply of the deficient neuropeptide or a neuropeptideoffering an equivalent mode of action.

Our data demonstrates that TRPV1⁺ sensory afferents control pancreatictissue access for immune cells, which may occur through modifying theirimmigration, residence, emigration or a combination of these elements.It is likely that progressive islet infiltration will also compoundβ-cell stress, which we believe is central to T1D, including attractionof autoreactive T cell pools. There is human disease precedence for arole of sensory neurons controlling lymphocyte tissue access, since rarepatients without sensory nerves (CIPA syndrome) succumb to massiveinfections with little tissue infiltration, despite normal in vitroimmune functions (Indo et al., 1996).

We discovered mutations in the coding sequence of TRPV1^(NOD) genecontained within the Idd4 diabetes risk locus (Grattan et al., 2002;Ivakine et al., 2005; McAleer et al., 1995). NOD.B6.Idd4 congenic miceshow normalized behavioral, electrophysiological and insulin-resistancephenotypes. Intriguingly, the TRPV1 locus is contained within otheroverlapping autoimmune loci (eae7, orch3, streptozotocin sensitivity)(Babaya et al., 2005; Butterfield et al., 1999; Butterfield et al.,1998), raising the possibility that TRPV1 may play a role in otherautoimmune conditions. Indeed, B6 mice, relatively resistant tostreptozotocin-induced T1D, show increased diabetes susceptibility inB6.TRPV1^(−/−) mice (data not shown).

In conclusion, our collective findings identify TRPV1⁺ sensory neuronsas important elements of diabetes pathoetiology, with effects that aresuggestive of possible mechanisms of the tissue-selectivity of thedisease, its links to p-cell physiology, stress and insulin resistance.Our observations open the possibility that sensory nerve dysfunction maycontribute to prediabetes initiation and progression in diabetes-pronehumans.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementherein described and shown. It will be apparent to those skilled in theart that various changes may be made without departing from the scope ofthe invention and the invention is not to be considered limited to whatis shown and described in the specification and any drawings/figuresincluded herein.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objectives and obtain theends and advantages mentioned, as well as those inherent therein. Theembodiments, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1-6. (canceled)
 7. A method comprising the following steps: (a)identifying a mammal having any one or more of the following:hyperinsulinism, insulin deficiency, high blood glucose levels, abnormalinsulin resistance, glucose intolerance, pancreatic islet inflammation,or pancreatic islet infiltration of T cells; and (b) administering tosaid mammal a composition comprising a sensory afferent neuronneuropeptide.
 8. The method of claim 7, wherein said mammal has diabetesor prediabetes.
 9. The method of claim 8, wherein said mammal has Type 1diabetes (T1D).
 10. The method of claim 8, wherein said mammal has Type2 diabetes (T2D).
 11. The method of claim 7, wherein said mammal is ahuman.
 12. The method of claim 7, wherein said sensory afferent neuronneuropeptide is substance P (sP).
 13. The method of claim 7, whereinsaid composition is administered in step (b) via intra-arterial (i.a.)injection.
 14. The method of claim 7, wherein said composition comprisesan amount of said sensory afferent neuron neuropeptide effective toreduce insulin resistance or enhance insulin sensitivity in said mammal.15. The method of claim 7, wherein said composition comprises an amountof said sensory afferent neuron neuropeptide effective to reduce ornormalize blood glucose levels in said mammal.
 16. The method of claim7, wherein said composition comprises an amount of said sensory afferentneuron neuropeptide effective to reduce inflammation or T cellinfiltration in the pancreatic islet of said mammal.
 17. A methodcomprising the following steps: (a) identifying a mammal having any oneor more of the following: hyperinsulinism, insulin deficiency, highblood glucose levels, abnormal insulin resistance, glucose intolerance,pancreatic islet inflammation, or pancreatic islet infiltration of Tcells; and (b) administering to said mammal a composition comprising acapsaicinoid compound.
 18. The method of claim 17, wherein said mammalhas diabetes or prediabetes.
 19. The method of claim 18, wherein saidmammal has Type 1 diabetes (T1D).
 20. The method of claim 18, whereinsaid mammal has Type 2 diabetes (T2D).
 21. The method of claim 17,wherein said mammal is a human.
 22. The method of claim 17, wherein saidcapsaicinoid compound is capsaicin.
 23. The method of claim 17, whereinsaid composition is administered in step (b) via intra-arterial (i.a.)injection.
 24. The method of claim 17, wherein said compositioncomprises an amount of said capsaicinoid compound effective to reduceinsulin resistance or enhance insulin sensitivity in said mammal. 25.The method of claim 17, wherein said composition comprises an amount ofsaid capsaicinoid compound effective to reduce or normalize bloodglucose levels in said mammal.
 26. The method of claim 17, wherein saidcomposition comprises an amount of said capsaicinoid compound effectiveto reduce inflammation or T cell infiltration in the pancreatic islet ofsaid mammal.